CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 2, March 2012
AFRICA
99
methods, the effects of experimental malaria on blood pressure
mechanisms, with a view to further understanding its role with
regard to blood pressure changes.
Methods
Five seven-week-old male Wistar rats weighing 150–180 g and
six Swiss mice weighing 30 g were obtained and kept at the
animal house of the Faculty for the study. The animals were kept
at a room temperature of 27
±
2°C with 12-h light/dark cycles.
They were fed with standard rat food and water
ad libitum
.
Approval for the study was sought and obtained from the Faculty
Ethics and Animal Regulations Committee.
The Swiss mice were used in the induction, maintenance
and preservation of the malaria parasite model (
Plasmodium
berghei
), which cannot be maintained in the Wistar rats used for
the vascular tissue studies.
28-31
Parasitaemia was maintained in the mice, using
Plasmodium
berghei
for the animal model. Briefly, parasitic infection was
induced by intraperitoneal injection of 4
×
10
6
(0.4 ml of para-
sitised blood in phosphate-buffered saline)
Plasmodium berghei
parasites. Development of parasitaemia in the infected mice was
monitored by microscopic examination of a blood film (Giemsa-
stained thin blood films) from the infected mice. On the fourth
day post-inoculation, blood pressure and heart rates were meas-
ured in the infected and control mice.
All mice were anaesthetised by intraperitoneal injection of
sodium thiopentone (50 mg/kg body weight). A polyethylene
catheter was inserted into the right jugular vein and another
into the left carotid aorta and connected to a pressure trans-
ducer (Statham P23XL) and Ugo Basile Polygraph (Model 7050,
Varese, Italy) for blood pressure and heart rate (HR) measure-
ments. Heparin (500 IU/kg) (Upjohn) was injected to prevent
intravascular blood clotting.
The animals were allowed to stabilise for at least 30 min
before recording. The blood pressure was recorded at a chart
speed of 10 mm/s and the heart rate was measured by increasing
the chart speed on the machine to 50 mm/min. The mean arterial
pressure (MAP) was calculated as the sum of the diastolic pres-
sure and 1/3 pulse pressure.
The thoracic aorta of the rats was rapidly dissected out and
placed in ice-cold, oxygenated, modified physiological saline
solution (PSS) with the following composition (mM): NaCl 119,
KCl 4.7, CaCl
2
2.5, MgSO
4
· H
2
O 1.2, KH
2
PO
4
1.2, NaHCO
3
24.9,
and glucose 11.1, pH 7.4. It was then cleaned of loosely adher-
ing fat and connective tissue and cut into ~2-mm rings. Each
aortic ring was suspended in an organ bath containing 20 ml of
well-oxygenated (95% O
2
, 5% CO
2
) PSS at 37°C. The rings were
allowed 90 min to equilibrate before the commencement of the
various protocols.
Force generation was monitored by means of an isometric
transducer (Grass model FT.03 isometric transducer) connected
to a Grass multichannel polygraph (Model 79D, Grass, Quincy,
MA, USA). The resting tension in the aortic rings was adjusted
to 1.0 g, which was found to be the optimal tension for inducing
a maximal contraction in preliminary experiments. The aortic
strips were first contracted with 80 mM KCl and this response
was taken as 100%.
Contractile responses were each expressed as a percentage
of the contraction previously induced by 80 mM KCl. Dose–
response tests to phenylephrine were carried out by cumulative
addition of the agonist to the bath in the presence or absence of
parasitised blood pre-incubated for 10 min.
The relaxation responses to acetylcholine were assessed
cumulatively in rings pre-contracted with 10
-6
M (EC
70
) phenyle-
phrine (PE) in the presence or absence of parasitised blood. The
magnitude of relaxation was compared with the pre-contraction
induced by phenylephrine.
32
Statistical analysis
Results are presented as means
±
SEM and comparison of the
means was done using the Student’s
t
-test. A
p
-value
<
0.05
was considered statistically significant. Contractile responses
are expressed as percentage (%) of maximal response to 80
mM KCl. The concentration–response curves for acetylcholine
were constructed using computer software from Origin™ 5.0
(Microcal Software Inc, Northampton, USA), and EC
50
and
EC
70
values (concentrations producing 50 and 70% of maximum
responses) were determined graphically.
Results
The arterial blood pressure, pulse pressure and heart rates of
control and malaria-parasitised rats are presented in Fig. 1. Mean
arterial pressure was significantly (
p
<
0.05) reduced in the para-
sitised (100
±
12 mmHg) rats when compared with controls (125
±
10 mmHg). Pulse pressure was also significantly (
p
<
0.05)
reduced in the parasitised rats (15
±
5 mmHg), compared with
controls (23
±
3 mmHg) (Fig. 2). Malaria parasitaemia did not
significantly alter heart rate (Fig. 3).
The dose–response curves for phenylephrine are presented in
Fig. 4. Parasitaemia resulted in a significant (
p
<
0.05) enhance-
ment (leftward shift) of the phenylephrine dose–response curve.
The mean EC
70
values for phenylephrine contractions in the
various ring preparations were 7
×
10
-7
M for the control and
Fig. 1. Mean arterial pressure in control and malaria-
parasitised rats.
150
100
50
0
Blood pressure (mmHg)
control
malaria group
*
p
<
0.05
*
Fig. 2. Pulse pressure in control and malaria-parasitised
rats.
30
20
10
0
Blood pressure (mmHg)
control
malaria group
*
p
<
0.05
*
